Acetylcholinesterase



 Acetylcholinesterase (AChE) is key enzyme in the nervous system of animals. By rapid hydrolysis of the neurotransmitter, acetylcholine (ACh), AChE terminates neurotransmission at cholinergic synapses. It is a very fast enzyme, especially for a serine hydrolase, functioning at a rate approaching that of a diffusion-controlled reaction. AChE inhibitors are among the key drugs approved by the FDA for management of Alzheimer's disease (AD). The powerful toxicity of organophosphorus (OP) poisons is attributed primarily to their potent AChE inhibitors.



Key Enzyme in the Nervous System


Solution of the three-dimensional (3D) structure of Torpedo californica acetylcholinesterase (TcAChE) in 1991 opened up new horizons in research on an enzyme that had already been the subject of intensive investigation. The unanticipated structure of this extremely rapid enzyme, in which the active site was found to be buried at the bottom of a deep and narrow gorge, lined by 14 aromatic residues (colored dark magenta) , led to a revision of the views then held concerning substrate traffic, recognition and hydrolysis. To understand how those aromatic residues behave with the enzyme, see Flexibility of aromatic residues in acetylcholinesterase. Solution of the 3D structure of acetylcholinesterase led to a series of theoretical and experimental studies, which took advantage of recent advances in theoretical techniques for treatment of proteins, such as molecular dynamics and electrostatics and to site-directed mutagenesis, utilizing suitable expression systems. [Acetylcholinesterase hydrolysizes the neurotransmitter acetylcholine (ACh), producing choline and an acetate group. ACh directly binds Ser200 (via its nucleophilic Oγ atom) within the catalytic triad (Ser200, His440, and Glu327) (ACh/TcAChE structure [[2ace]]). The residues Trp84 and Phe330 are also important in the ligand recognition . After this binding acetylcholinesterase hydrolysizes ACh. See also: AChE inhibitors and substrates

Treatment of Alzheimer's disease
Alzheimer's disease (AD) is a disorder that attacks the central nervous system through progressive degeneration of its neurons. AD occurs in around 10% of the elderly and, as yet, there is no known cure. Patients with this disease develop dementia which becomes more severe as the disease progresses. It was suggested that symptoms of AD are caused by decrease of activity of cholinergic neocortical and hippocampal neurons. Treatment of AD by ACh precursors and cholinergic agonists was ineffective or caused severe side effects. ACh hydrolysis by AChE causes termination of cholinergic neurotransmission. Therefore, compounds which inhibit AChE might significantly increase the levels of ACh depleted in AD. Indeed, it was shown that AChE inhibitors improve the cognitive abilities of AD patients at early stages of the disease development.



The first generation of AD drugs - monovalent AChE inhibitors
The first generation of AD drugs were AChE inhibitors: alcaloids like (-)-Huperzine A (HupA) and (-)-galanthamine (GAL, Reminyl); synthetic compounds tacrine (Cognex) and rivastigmine (Exelon).

HupA
HupA, discovered by Chinese scientists from 1980s, has been proved to be a powerful, highly specific, and reversible inhibitor of AChE. The crystal structure of the complex of TcAChE with HupA at 2.5 Å resolution (1vot) was determined in 1997 and it shows an unexpected orientation for the inhibitor with surprisingly few strong direct interactions with protein residues to explain its high affinity. HupA binds to TcAChE at the active site, and its observed orientation is almost orthogonal in comparison to ACh. The principal interactions of HupA with TcAChE are including: a direct <scene name='1vot/1vot_199_130_117/2'>hydrogen bond with Tyr130 and HBs with Glu199 and Gly117 <font color='orange'>(colored orange) through a water molecule as a linker at the bottom of the gorge; cation-π interactions between the amino group of <scene name='1vot/1vot_84_330/2'>HupA and Trp84 and Phe330 <font color='lime'>(colored lime) with the distance between the nitrogen and the centroid of the aromatic rings of 4.8 and 4.7 Å, respectively; at the top of the gorge, hydrogen bonds through two water molecules as linkers formed between the amino group of <scene name='1vot/1vot_70_72_81_85_121/3'>HupA and Tyr70, Asp72, Ser81, Asn85 and Tyr121 <font color='magenta'>(colored magenta). An unusually short (~3.0 Å) C-H→O HB has been seen between the ethylidene methyl group of <scene name='1vot/1vot_440/2'>HupA and the main chain oxygen of His440 <font color='crimson'>(colored crimson).

Galanthamine
<scene name='AChE_inhibitors_and_substrates/Com_view_gal/1'>Galanthamine (GAL, Reminyl). <scene name='AChE_inhibitors_and_substrates/Com_view_gal/2'>GAL <font color='red'>(red) is an alkaloid from the flower snowdrop (Galanthus nivalis). The X-ray crystal structure of the TcAChE/GAL complex (1dx6) was determined at 2.3 Å resolution. The inhibitor binds at the base of the active site gorge of TcAChE, interacting with both the choline-binding site (Trp84) and the acyl-binding pocket (Phe288, Phe290). The tertiary amine appears to make a non-conventional hydrogen bond, via its N-methyl group, to Asp72. The hydroxyl group of the inhibitor makes a strong hydrogen bond (2.7 Å) with Glu199. <font color='gray'>ACh (gray) is shown for comparison.

Tacrine
<scene name='AChE_inhibitors_and_substrates/Com_view_tacrine/1'>Tacrine (Cognex). In the X-ray crystal structure of TcAChE/<scene name='AChE_inhibitors_and_substrates/Com_view_tacrine/2'>tacrine complex which was determined at 2.8 Å resolution, the tacrine is seen <font color='magenta'>(magenta) bound in the active site of TcAChE (1acj). <font color='gray'>ACh (gray) is shown for comparison.

Rivastigmine
<scene name='1gqr/Com_view/1'>Rivastigmine (Exelon) is a carbamate inhibitor of AChE, and it is currenly used in therapy of Alzheimer's Disease. Rivastigmine (colored yellow) interacts with TcAChE <font color='lime'>(colored lime) at the <scene name='1gqr/Active_site/4'>active-site gorge (1gqr). The carbamyl moiety of rivastigmine is <scene name='1gqr/Active_site/9'>covalently bound to the active-site S200 Oγ. The second part of rivastigmine (the leaving group), NAP ((−)-S-3-[1-(dimethylamino)ethyl]phenol) is also held in the active-site gorge, but it is <scene name='1gqr/Active_site/6'>separated from the carbamyl moiety, hence, carbamylation took place. The <scene name='1gqr/Active_site/7'>crystal structure of TcAChE/<font color='magenta'>NAP (colored magenta) is known (1gqs). The <font color='violet'>TcAChE active-site residues which are interacting with NAP are <font color='violet'>colored violet. NAP is located in a similar region of TcAChE active site, but with different orientation than that of the NAP part (colored yellow) in the TcAChE/rivastigmine complex. Only H440 and F330 significantly change their side-chain conformations. <scene name='1gqr/Active_site/8'>Overlap of the TcAChE active sites in 4 different structures (<font color='lime'>TcAChE /rivastigmine (1gqr), <font color='violet'>TcAChE /<font color='magenta'>NAP (1gqs), <font color='cyan'>native TcAChE (2ace), and TcAChE/VX (1vxr, TcAChE colored white and VX black) reveals that the conformation of H440 in the TcAChE/NAP structure is very similar its conformation in the native TcAChE (2ace), but the distance between H440 Nδ and E327 Oε is significantly longer in the TcAChE/rivastigmine and the TcAChE/VX complexes. This structural change disrupts the catalytic triad consisting of S200, E327, H440. This could explain the very slow kinetics of AChE reactivation after its inhibition by rivastigmine.

The second generation of AD drugs - bivalent AChE inhibitors
The active site of <scene name='1zgb/Com_view/1'>TcAChE consists of <scene name='1zgb/Act_site/3'>two binding subsites. First of them is the "catalytic anionic site" (CAS), which involves mentioned above catalytic triad <scene name='1zgb/Act_site/8'>Ser200, His440, and Glu327 <font color='orange'>(colored orange) and the conserved residues <scene name='1zgb/Act_site/5'>Trp84 and <scene name='1zgb/Act_site/10'>Phe330 also participating in ligands recognition. Another conserved residue <scene name='1zgb/Act_site/11'>Trp279 <font color='cyan'>(colored cyan) is situated at the second binding subsite, termed the "peripheral anionic site" (PAS), ~14 Å from CAS. Therefore, the ligands that will be able to interact with both these subsites, will be more potent AChE inhibitors in comparison to compounds interacting only with CAS (mentioned in the previous section "The first generation of AD drugs - monovalent AChE inhibitors"). One of the ways to produce such ligands is to introduce two active substances into one compound. If it is spatially necessary these subunits could be divided by alkyl linker with suitable length. For example, according to the strategy of the use of a bivalent ligand, the <scene name='1zgb/Comp/7'>inhibitor (RS)-(±)-tacrine-(10)-hupyridone ((R)-3 or (S)-3) was designed and synthesized. It consists of mentioned above <scene name='1zgb/Comp/8'>tacrine <font color='magenta'>(colored magenta), 10-carbon <scene name='1zgb/Comp/9'>linker <font color='yellow'>(yellow) , and <scene name='1zgb/Comp/10'>hupyridone <font color='red'>(red). The tacrine moiety of this inhibitor binds at the CAS, the linker spans the <scene name='1zgb/Act_site/12'>active-site gorge, and the hupyridone moiety binds at the PAS (1zgb). There are also only PAS-binding AChE inhibitors, <scene name='2j3q/Active_site/6'>Thioflavin T <font color='magenta'>(magenta) is a good example of them. <scene name='2j3q/Active_site/7'>Superposition of the crystal structure of the <font color='red'>edrophonium /TcAChE (CAS-binding inhibitor) (2ack) on the <font color='magenta'>thioflavin T /TcAChE complex structure (2j3q) shows that these ligands' positions do not overlap. Of note is that Phe330, which is part of the CAS, is the single residue interacting with <font color='magenta'>thioflavin T. This residue is the only one which significantly <scene name='2j3q/Active_site/9'>changes its conformation to avoid clashes in comparison to other CAS residues of the <font color='red'>edrophonium /TcAChE complex.

Compound 3
Described above, <scene name='1w4l/Al/1'>Galantamine (abbreviated as <scene name='1w4l/Al/2'>GAL ; <font color='red'>colored red ) is a CAS-binding inhibitor and it is currently used in therapy of Alzheimer's Disease under the trade name Razadyne. Conjugate of GAL through the <scene name='1w4l/Al/3'>alkyl linker (8 carbons, <font color='black'>yellow ) with a <scene name='1w4l/Al/4'>phthalimido moiety <font color='blueviolet'>(blueviolet) called compound 3 has a larger affinity for AChE than that of GAL alone. This is similar to previously described cases of bivalent ligands (e.g. (RS)-(±)-tacrine-(10)-hupyridone). A comparison between <scene name='1w4l/Comparison/1'>compound 3 /TcAChE (1w4l) and <scene name='1w4l/Comparison/2'>galanthamine/TcAChE structure (1dx6) shows an identical binding mode of the <font color='red'>GAL-moiety (transparent red) of compound 3 to that of <font color='blue'>GAL alone (blue) at the CAS. A <font color='gray'>PEG molecule (gray) is located at the active site of the galanthamine/TcAChE structure. The alkyl linker spans the active-site gorge and the phthalimido moiety of compound 3 is situated near Trp279 at the PAS. Compound 3 has higher affinity to TcAChE than GAL. This can be explained by the higher number of interactions between compound 3 (which interacts not only with residues within CAS but also within PAS) and TcAChE relative to GAL.

Aricept (donepezil, E2020)
<scene name='Main_Page/E2020_in_ache_spinning/1'>Aricept (E2020) (Donepezil) is one of the most interesting drugs that have been designed as AChE bivalent inhibitors. It was developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies prior to elucidation of the 3D structure of Torpedo californica AChE (TcAChE) (1ea5). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range. The X-ray structure of the E2020-TcAChE complex (1eve) shows that E2020 has a <scene name='1eve/E2020_close_up_with_84_279/13'>unique orientation along the active-site gorge, extending from the anionic subsite (<scene name='1eve/E2020_close_up_with_84lbld/7'>W84 ) of the active site, at the bottom, to the peripheral anionic site (<scene name='1eve/E2020_close_up_with_84_279lbld/5'>near W279 ), at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole' but only <scene name='1eve/E20_interactionshown/8'>indirectly via solvent molecules. The X-ray structure shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE, to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.

</StructureSection>

Organophosphorus acid anhydride nerve agents
<applet load='Soman1.pdb' size='500' frame='true' align='right' scene='2wfz/Al/1' />

Organophosphorus (OP) acid anhydride nerve agents are potent inhibitors which rapidly phosphonylate AChE and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. As was mentioned above, AChE hydrolysizes the neurotransmitter <scene name='2wfz/Al/2'>ACh, producing <scene name='2wfz/Al/3'>choline and an acetate group. <scene name='2wfz/Al/2'>ACh directly binds catalytic <scene name='2wfz/Al/4'>Ser200 (via its nucleophilic Oγ atom). <scene name='2wfz/Al/5'>Soman, O-(1,2,2-trimethylpropyl) methylphosphonofluoridate (<font color='violet'>fluorine atom is colored violet and <font color='darkmagenta'>phosphorus atom is colored darkmagenta ), is one of the most toxic OPs. Soman inhibits AChE by <scene name='2wfz/Al/6'>covalent binding to catalytic Ser200, <scene name='2wfz/Al/7'>mimicking ACh. This process <scene name='2wfz/Al/8'>(phosphonylation) implicates nucleophilic attack of the Ser200 nucleophilic Oγ atom on the phosphorus atom of soman, with concomitant departure of its fluoride atom. After that AChE catalyzes the <scene name='2wfz/Al/9'>dealkylation ("aging") of the soman or other OP. This causes irreversible inhibition of AChE, "aged" soman/AChE conjugate can not be reactivated. However, before “aging”, at the step of <scene name='2wfz/Al/8'>phosphonylation, AChE can be <scene name='2wfz/Al/11'>reactivated by nucleophiles, such as pralidoxime (2-PAM), resulting in <scene name='2wfz/Al/12'>cleavage of the phosphonyl adduct from Ser200 Oγ. At the <scene name='2wfz/Ali/3'>active site of the nonaged soman/TcAChE conjugate (2wfz) the catalytic His440 forms hydrogen bonds with Ser200 Oγ and Glu327 Oε1 via its Nε2 and Nδ1 nitrogens, respectively. The O2 atom of soman is within hydrogen bonding distance of His440 Nε2. Soman O1 mimicks carbonyl oxygen of ACh. A water molecule 1001 interacting with soman O2 is represented as a <font color='red'>red ball. The active site residues of the nonaged soman/TcAChE are colored <font color='yellow'>yellow. The O2 atom of the <scene name='2wfz/Ali/4'>dealkylated (aged) soman (2wg0) forms a salt bridge with His440 Nε2. The active site residues of the aged soman/TcAChE are colored <font color='pink'>pink. <scene name='2wfz/Ali/5'>Alignment of the structures of the nonaged (2wfz) and aged (2wg0) conjugates reveals a small, but important, change within the active site - the imidazole ring of His440 is tilted back to a native-like conformation after dealkylation. The water molecule 1001, which interacts with soman O2 in the nonaged crystal structure, is not within hydrogen bonding distance of O2 in the aged crystal structure. 2-PAM binds poorly to the nonaged phosphonylated enzyme (its electron density was not found) and binds in an <scene name='2wfz/Ali/7'>unfavorable and nonfunctional conformation after soman aging to TcAChE (2wg1).

3D Structures of AChE
Update June 2011

Acetylcholinesterase - AChE native
3lii – hAChE - recombinant human

1ea5, 2ace – TcAChE – trigonal – Torpedo californica

2j3d – TcAChE – monoclinic

1w75 – TcAChE – orthorhombic

1eea – TcAChE – cubic

2vt6, 2vt7 – TcAChE – different dosage

1qid to 1qim - TcAChE synchrotron radiation damage

1j06, 1maa – mAChE - mouse

1qo9 – DmAChE - Drosophila

1c2o, 1c2b – electrophorus AChE – Electric eel

AChE inhibitors (In Different Languages)
1eve AChE-Aricept complex, 1eve (Arabic), 1eve (Chinese), 1eve (Italian), 1eve (Russian), 1eve (Spanish), 1eve (Turkish)

1vot AChE-Huperzine A complex, 1vot (Chinese)

AChE active site inhibitors conjugating at the bottom of the active site gorge
2w9i – TcAChE + methylene blue

2wls – MosAChE + AMTS13

2vq6 – TcAChE + 2-PAM

2j3q – TcAChE + Thioflavin T

2ha0 – mAChE + ketoamyltrimethylammonium

2h9y – mAChE + TMTFA

1gpk, 1gpn, 1vot – TcAChE + huperzine

1gqr – TcAChE + rivastigmine

1gqs – TcAChE + NAP

1e66 – TcAChE + huprine

1dx4, 1qon – DmAChE + tacrine derivative

1oce – TcAChE + MF268

1ax9, 1ack – TcAChE + edrophonium 1amn – TcAChE + TMTFA

1acj – TcAChE + tacrine

1u65 – TcAChE + CPT-11

2bag - TcAChE + ganstigmine

2xi4 - TcAChE + aflatoxin

AChE peripheral site inhibitors conjugating at the surface of the protein
1ku6, 1mah - mAChE + fasciculin 2

1j07 - mAChE + decidium

1n5m - mAChE + gallamine

1n5r - mAChE + propidium

1b41, 1f8u - hAChE + fasciculin 2

1fss - TcAChE + fasciculin 2

2x8b - hAChE + fasciculin 2 + tabun

AChE bis inhibitors spanning the active site gorge
3i6m – TcAChE + N-piperidinopropyl galanthamine

3i6z - TcAChE + saccharinohexyl galanthamine

1zgb, 1zgc – TcAChE + tacrine (10) hupyridone

2w6c – TcAChE + bis-(-)-nor-meptazinol

2ckm, 2cmf – TcAChE + bis-tacrine

2cek – TcAChE + N-[8-(1,2,3,4-tetrahydroacridin-9-ylthio)octyl]-1,2,3,4-tetrahydroacridin-9-amine

1ut6 - TcAChE + N-9-(1,2,3,4-tetrahydroacridinyl)-1,8-diaminooctane

1odc - TcAChE + N-4-quinolyl-N-9-(1,2,3,4-tetrahydroacridinyl)-1,8-diaminooctane

1w4l, 1w6r, 1w76, 1dx6, 1qti - TcAChE + galanthamine and derivative

1q83, 1q84 - mAChE + TZ2PA6

1h22, 1h23 – TcAChE + bis-hupyridone

1hbj – TcAChE + quinoline derivativev

1e3q – TcAChE + bw284c51

1eve – TcAChE + e2020

1acl – TcAChE + decamethonium

AChE organophosphate inhibitors causing irreversible inhibition
2wu3 – mAChE + fenamiphos and HI-6

2wu4 – mAChE + fenamiphos and ortho-7

2jgf - mAChE + fenamiphos

2wfz, 2wg0, 2wg2, 1som - TcAChE + soman

2wg1 - TcAChE + soman + 2-PAM

2whp, 2whq, 2whr – mAChE + sarin and HI-6

2jgg - mAChE + sarin

2jgl - mAChE + VX and sarin

1cfj - TcAChE + sarin, GB

3dl4, 3dl7 – mAChE + tabun

2jey – mAChE + HLO-7

2c0p, 2c0q - mAChE + tabun

2jez - mAChE + tabun + HLO-7

2jf0 - mAChE + tabun + Ortho-7

2jgh - mAChE + VX

1vxo, 1vxr - TcAChE + VX

2jgi, 2jgm - mAChE + DFP

1dfp - TcAChE + DFP

2jgj, 2jgk, 2jge - mAChE + methamidophos

2gyu - mAChE + HI-6

2gyv - mAChE + Ortho-7

2gyw - mAChE + obidoxime

3gel - TcAChE + methyl paraoxon

AChE substrate analogues mimicking the binding of the substrate acetylcholine
2ha4 – mAChE (mutant) + acetylcholine

2vja, 2vjb, 2vjc, 2vjd, 2cf5 – TcAChE + 4-oxo-N,N,N-trimethylpentanaminium

2v96, 2v97, 2v98, 2v99 – TcAChE + 1-(2-nitrophenyl)-2,2,2-trifluoroethyl-arsenocholine

2ha2 – mAChE + succinylcholine

2ha3 - mAChE + choline

2ha5 – mAChE (mutant) + acetylthiocholine

2ha6 – mAChE (mutant) + succinylthiocholine

2ha7 – mAChE (mutant) + butyrylthiocholine

2ch4, 2c58 – TcAChE + acetylthiocholine

2c5g – TcAChE + thiocholine

2va9 - TcAChE + ‘caged’ arsenocholine

Others...
2j4f – TcAChE + Hg

1vzj – TcAChE tetramerization domain

1jjb – TcAChE + PEG

1qie, 1qif, 1qig, 1qih, 1qii, 1qij, 1qik – TcAChE synchrotron radiation damage

3m3d – TcAChE + Xe

Additional Resources
For additional information, see:

Alzheimer's Disease

AChE inhibitors and substrates

AChE inhibitors and substrates (Part II)

AChE inhibitors and substrates (Part III)

AChE bivalent inhibitors

AChE bivalent inhibitors (Part II)